Development and Validation of UV Spectrophotometric Method for ...

J Pharm Sci Bioscientific Res. 2016 6(3):365-370

ISSN NO. 2271-3681

Development and Validation of UV Spectrophotometric Method for Simultaneous Estimation of Cefuroxime Axetil and Linezolid in Tablet Dosage Form Rimakumari N. Chavda*, Hitendra N. Yadav, Madhuri A. Hinge, Shuchi P. Desai Department of Quality Assurance, ROFEL Shri G.M Bilakhia College of Pharmacy, Vapi, Gujarat, India

ABSTRACT: To develop simple, sensitive, accurate, rapid and precise first derivative spectrophotometric method for the simulataneous estimation of Cefuroxime Axetil (CEF) and Linezolid (LIN) in Tablet dosage form. The derivative spectrophotometric method was based on the determination of both the drugs at their respective zero crossing point (ZCP). The first order derivative spectrum was obtained in methanol and the determinations were made at 257.40 nm (ZCP of Linezolid) for Cefuroxime Axetil and 276.60 nm (ZCP of Cefuroxime Axetil) for Linezolid. The linearity was obtained in the concentration range 2-6 µg/ml for Cefuroxime Axetil and 2.4-7.2 µg/ml for Linezolid. The results of analysis have been validated statistically and by recovery studies. Key words: Cefuroxime Axetil (CEF), Linezolid (LIN), First Order Derivative Method. Article history:

INTRODUCTION:

DOS: DOR DOA Received 5 March 2016 Revised 13 March 2016 Accepted 20 March, 2016 Available online 01 May 2016

Cefuroxime Axetil is a bactericidal in action. Like other cephalosporins, the antibacterial activity of the drug results from inhibition of mucopeptide synthesis in the bacterial cell wall. That is active against Gram-negative bacteria. Cefuroxime Axetil is chemically (1 RS)-1-(acetyloxy)ethyl(6R,7R)-3[(carbamoyloxy)methyl]-7[[(Z)-2-(furan-2-yl)-2-(methoxyimino) acethyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate. Linezolid is a member of a new structural class of antibiotics, oxazolidinones. The oxazolidinones have a good activity against Gram-positive bacteria. They act uniquely by inhibiting the formulation of protein synthesis initiation in Gram-positive bacteria. Linezolid is chemically N-[(5S)-3-[3-fluoro-4-(4morpholinyl) phenyl]-2-oxo-5-oxazolidinyl] methyl]acetamide. Combination of Cefuroxime Axetil and Linezolid is used to treat bacterial infection.(1-5) The aim of the present work was to develop a new simple, rapid, selective method for the simultaneous determination of components having overlapping spectra in binary mixtures. The first order derivative method for simulataneous determinations were made at 257.40 nm (ZCP of LIN) for CEF and 276.60 nm (ZCP of CEF) for LIN.(6-16)

Citation: Chavda R. N., Yadav H. N., Hinge M. A., Desai S. P. Development and Validation of UV Spectrophotometric Method for Simultaneous Estimation of Cefuroxime Axetil and Linezolid in Tablet Dosage Form. J Pharm Sci Bioscientific Res. 2016 6(3): 365-370

*For Correspondence: Rimakumari N. Chavda Department of Quality Assurance,ROFEL Shri G.M Bilakhia College of Pharmacy, Vapi, Gujarat, India.

(www.jpsbr.org)

Figure 1: Structure of Cefuroxime Axetil

Chavda R. N. et al

Figure: 2 Structure of Linezolid

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MATERIALS AND METHODS: Instruments and Apparatus: A double beam UV-Visible spectrophotometer, SHIMADZU (model UV-1800, Software-UV Probe, Version 2.42) having two matched quartz cells with 1 cm light path. All weighing was done on Reptech electronic analytical balance. All the apparatus used were calibrated.

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2-6 µg/ml solutions of CEF were prepared in Methanol and spectrum was recorded between 200-400 nm. Spectrums for above concentration were obtained with n=5 and 2.4-7.2 µg/ml solutions of LIN were prepared in Methanol and spectrum was recorded between 200400nm. CEF at 257.40 nm(ZCP of LIN) and LIN at 276.44 nm(ZCP of CEF). The overlay spectrums of CEF and LIN at different concentration were recorded. Method:

Reagents and Chemicals:

Calibration curve for the CEF (2 - 6 µg/ml)

Cefuroxime Axetil (Gift sample, Wockhardt,Mumbai) and Linezolid (Gift sample, Aristo pharmaceuticals Pvt. Ltd,Mumbai). Methanol AR grade (Rankem)

Appropriate volume of aliquot from standard CEF stock solution was transferred to different volumetric flasks of 10 ml capacity. The volume was adjusted to the mark with the methanol to obtain concentration of 2, 3, 4, 5, and 6µg/ml. The curve of each solution against the Methanol was recorded. Absorbance of CEF at 257.40 nm (ZCP of LIN) was measured and the plot of absorbance vs. concentration was plotted.

Marketed Formulation: Linox*-XT tablet manufactured by Unichem laboratories Ltd. Preparation of Standard solution:

Calibration curve for the LIN (2.4 - 7.2 µg/ml) Cefuroxime Axetil (CEF) standard stock solution: Accurately weighed quantity of CEF 100 mg was transferred to 100ml volumetric flask, dissolved and diluted up to mark with Methanol to give a standard stock solution having strength 1000µg/ml. Then pipette out 10ml from the standard stock solution in other 100ml volumetric flask and diluted up to mark with Methanol to give a working standard solution having strength 100µg/ml. Pipette out 25ml from the working standard solution in other 50ml volumetric flask and diluted up to mark with Methanol to give a working standard solution having strength 50µg/ml. Linezolid (LIN) standard stock solution: Accurately weighed quantity of LIN 100 mg was transferred into 100 ml volumetric flask, dissolved and diluted up to mark with Methanol to give a stock solution having strength 1000µg/ml. Then pipette out 10ml from the standard stock solution in other 100ml volumetric flask and diluted up to mark with Methanol to give a stock solution having strength 100µg/ml. Pipette out 25ml from the working standard solution in other 50ml volumetric flask and diluted up to mark with Methanol to give a working standard solution having strength 50µg/ml.

Appropriate volume of aliquot from standard LIN stock solution was transferred to different volumetric flasks of 10 ml capacity. The volume was adjusted to the mark with the methanol to obtain concentration of 2.4, 3.6, 4.8, 6, and 7.2µg/ml. The curve of each solution against the Methanol was recorded. Absorbance of LIN at 276.60 nm (ZCP of CEF) was measured and the plot of absorbance vs. concentration was plotted. Preparation of Sample solution: Take twenty tablets were weighed and finely powdered. The powder equivalent to 500 mg CEF and 600 mg LIN was accurately weighed and transferred to volumetric flask of 100ml capacity. Powder was dissolved in methanol in volumetric flask. The flask was shaken and volume was made up to the mark with methanol. The solution was filtered through whatmann filter paper. Then 10 ml of solution was taken and transferred to volumetric flask of 100 ml and volume was made up to the mark with methanol. Further 0.4 ml of this solution was transferred to volumetric flask of 10ml capacity. Volume was made up to the mark with methanol to give a solution containing 4μg/ml CEF and 4.8μg/ml LIN. This solution was used for the estimation of CEF and LIN in tablet dosage form.

Selection of Analytical Wavelength:

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Estimation of CEF and LIN by First order derivative Method. Absorbance of the resulting solution was measured at 257.40 nm for determination of CEF (ZCP of LIN) and at 276.60 nm for determination of LIN (ZCP of CEF).

ISSN NO. 2271-3681

6. RESULT AND DISCUSSION: The Linearity range for CEF and LIN was found to be in the range of 2-6 µg/ml and 2.4-7.2 µg/ml respectively. The method was determined at five concentration levels for CEF at 257.40 nm (ZCP of LIN) and LIN at 276.60 nm (ZCP of CEF) independently.

Method Validation: 1. Accuracy Accuracy was determined by calculating recovery of CEF and LIN by the standard addition method. Known amounts of standard solutions of CEF and LIN were added to test solutions. The recovery was calculated by measuring absorbance. 2. Precision The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogenous samples.

ZCP of CEF 276.60 nm

Figure: 3 Overlay Spectra of CEF at (2-6 µg/ml)

3. Repeatability Standard solutions of CEF and LIN were prepared of linearity range and spectrums were recorded. Absorbance was measured at 257.40 nm and 276.60 nm. 4. Intraday and Inter day Precision Variations of results within the same day (intraday), variation of results between days (inter day) were analyzed. Intraday precision was determined by analyzing CEF and LIN individually for three times in the same day at 257.40 nm and 276.60 nm.

ZCP of LIN 257.40 nm

Figure: 4 Overlay Spectra of LIN at (2.4-7.2 µg/ml)

Inter day precision was determined by analyzing CEF and LIN individually daily for three day at 257.40 nm and 276.60 nm ZCP of CEF 276.60 nm

5. Linearity The linearity of analytical method is its ability to test results that are directly proportional to the concentration of analyte in sample within a given range. The range of analytical method is the interval between the upper and lower levels of analyte that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity.

Chavda R. N. et al

ZCP of LIN 257.40 nm

Figure: 5 Overlay spectra of CEF and LIN

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ISSN NO. 2271-3681

Table: 1 Linearity data for CEF at 257.40 nm (ZCP of LIN) Concentration (µg/ml) 2 3 4 5 6

Absorbance at 257.40 nm Mean± S.D. (n=5) 0.0050 ± 0.00010 0.0069 ± 0.00012 0.0087 ± 0.00016 0.0108 ± 0.00016 0.0128 ± 0.00016

Drugs Conc.

Absorbance

%RSD

(µg/ml) Mean ± S.D. (n=3) CEF

LIN

0.014

3

0.0070 ± 0.00010

1.428

4

0.0088 ± 0.00015

1.732

5

0.0111 ± 0.00010

0.900

3.6

-0.0119 ± 0.00010

0.840

4.8

-0.0164 ± 0.00017

1.054

6

-0.0188 ± 0.00011

0.609

0.012 y = 0.0020x + 0.0010 R² = 0.9999

Abs

0.01

Table: 4 Interday precision

0.008 0.006

Drugs Conc.

0.004

Absorbance

%RSD

(µg/ml) Mean ± S.D. (n=3)

0.002 CEF

0 0

2

4 Conc

6

8

Figure: 6 Calibration curve of CEF at 257.40 nm LIN Table: 2 Linearity data for LIN at 276.60 nm (ZCP of CEF) Concentration (µg/ml) 2.4 3.6 4.8 6 7.2

Absorbance at 223.38 nm Mean ± S.D. (n=5) 0.0079 ± 0.00012 0.0116 ± 0.00016 0.0157 ± 0.00026 0.0182 ± 0.00030 0.0228 ± 0.00040

3

0.0069 ± 0.00011

1.658

4

0.0089 ± 0.00017

1.943

5

0.0111 ± 0.00017

1.558

3.6

-0.0118 ± 0.00020

1.600

4.8

-0.0162 ± 0.00025

1.549

6

-0.0188 ± 0.00015

0.812

Table: 5 Accuracy study for CEF and LIN Drugs Level Amt. of Amt. of Total Amt. Sample STD

Amt. Found

(µg/ml) Spiked

(µg/ml)

Conc

0 0

5

10

CEF

-0.005 Abs

% Recovery

-0.01

y = -0.0031x - 0.0008 R² = 0.9978

-0.015

LIN

-0.02 -0.025

Figure: 7 Calibration curve of LIN at 276.60 nm

80

4

3.2

7.2

7.30

101.38

100

4

4

8

7.91

98.95

120

4

4.8

8.8

8.66

98.48

80

4.8

3.8

8.6

8.55

99.06

100

4.8

4.8

9.6

9.58

99.79

120

4.8

5.7

10.5 10.60

100.38

Table: 6 Analysis of marketed formulation

Table: 3 Intraday precision

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Formulation Actual

Amt.

(Tablet

obtained

Linox*-XT)

Batch No.

conc. (µg/ml)

(µg/ml)

CEF

LIN

CEF

4

4.8

4.10 4.72

MCRAE03

ISSN NO. 2271-3681

%CEF ±

%LIN ±

SD

SD

(n=3)

(n=3)

LIN 101.5± 98.33± 0.020

0.010

CONCLUSION: From the overlay spectra of Cefuroxime Axetil and Linezolid it is observed that estimation of both the drug can be possible using First order derivative method. The method was developed and validated. The value of % recovery and standard deviation reveals that the proposed method was successfully utilized for the estimation of Cefuroxime Axetil and Linezolid in tablet dosage form. A simple, rapid and sensitive method is proposed for the analysis of two binary mixtures with overlapping spectra. The method involves the generation of absorbance spectra followed by measurement of the absorbance. Therefore, the presented methodology is adequate for the routine quality control analysis of these fixed-dose combinations. ACKNOWLEDGEMENT: The authors are thankful to the Wockhardt, Mumbai for providing the gift sample of Cefuroxime Axetil and Aristo Pharmaceuticals Pvt. Ltd, Mumbai for providing gift sample of Linezolid. The authors are also thankful to the Principle, Rofel Shri G. M. Bilakhia College of Pharmacy,Vapi, India, for providing the required facilities to carry out this research work. REFERENCES: 1) Backett AH., and Stenlake JB. Practical pharmaceutical chemistrypart II, CBS publishers and distributors, Delhi,Fourth Edition.2002:264-337. 2) Chatwal GR., and Anand S. Instrumental Methods of Chemical Analysis, Himalaya Publishing House, Fifth Edition.2012:180-198. 3) Skoog DA., Holler FJ., and Nieman TA. Introduction to UV Spectroscopy; Principle of instrumental analysis,Thomson Brooks-Cole publication, Fifth Edition.2004:301- 305.

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